Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-α production by mononuclear phagocytes

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1 Nrf2 mediates induced CLEC5A and TNFα Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNFα production by mononuclear phagocytes YiLin Cheng 1,2, YeeShin Lin 1,2,3, ChiaLing Chen 4, TsungTing Tsai 5, ChengChieh Tsai 6, YanWei Wu 1,2, YiDan Ou 3, YuYi Chu 7, JuMing Wang 1,2,7, ChiaYi Yu 3, and ChiouFeng Lin 2,5,8 * 1 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; 2 Center of Infectious Diseases and Signaling Research, National Cheng Kung University, Tainan 701, Taiwan; 3 Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; 4 Translational Research Center, Taipei Medical University, Taipei 110, Taiwan; 5 Department of Microbiology and Immunology, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; 6 Department of Nursing, Chung Hwa University of Medical Technology, Tainan 717, Taiwan; 7 Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan 701, Taiwan; 8 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan Supplemental figure legends Figure S1 Protein assay for Figure 1. Figure S2 Protein assay for Figure 2. Figure S3 Protein assay for Figure 3. Figure S4 Protein assay for Figure 4. Figure S5 Protein assay for Figure 5. 1

2 Nrf2 mediates induced CLEC5A and TNFα Figure S6 Impact of NS2B3, NS3 or GFPFlag on ER stress, Nrf2 activation and CLEC5A expression. RAW264.7 cells were transfected with pcr3.1 vector (vector), pcr3.1ns2b3flag (NS2B3), or pcr3.1ns3flag (NS3). (A) Western blot analysis showed the expression of Flag. (B) ARE activity assay was performed to illustrate Nrf2 activation. (C) Confocal immunostaining was used to detect nuclear translocation of Nrf2 (green) in transfected cells. DAPI was used as a nuclear stain (blue). RAW264.7 cells were transfected with GFPFlag or NS2B3Flag. Western blot analysis showed the expression of Flag (D), phosphorylated PERK Thr981, PERK, and CLEC5A (E). The relative protein expression was determined by the ratio of the detected proteins to an internal βactin control. (F) Flow cytometric analysis of the surface expression of CLEC5A in NS2B3transfected RAW264.7 cells. The data are shown as the mean fluorescent intensity. (G) Confocal immunostaining was used to detect nuclear translocation of Nrf2 (green) in transfected cells. DAPI was used for nuclear staining (blue). For all quantified data, values are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01, compared with vector or GFPFlag. ns, not significant. Figure S7 Protein assay for Figure 6. 2

3 Cheng et al. Supplemental Figure 1 For Figure 1A For Figure 1B NS βactin Nrf2 βactin (MOI) shluc shnrf2

4 Cheng et al. Supplemental Figure 2 For Figure 2B pperk PERK IRE1 α ATF6 (Proform) ATF6 (Cleaved form) CHOP (h)

5 Cheng et al. Supplemental Figure 3 26 For Figure 3A 26 For Figure 3C 26 For Figure 3D NS3 NS Scremble sirna Nrf2 sirna Nrf ATRA DMSO For Figure 3E PBA GSK (h) PBA GSK 26 4PBA GSK

6 Cheng et al. Supplemental Figure 4 For Figure 4B Scramble sirna CLEC5A sirna

7 Cheng et al. Supplemental Figure 5 For Figure 5A For Figure 5B Flag pperk PERK 26 Flag Vector NS2B Vector NS2B3

8 Cheng et al. Supplemental Figure 6 A B C Flag Vector NS2B3 NS3 Relative ARE Activity (Fold increase) Vector D E F G Flag pperk PERK CLEC5A βactin βactin βactin * NS2B ns NS3 Vector NS2B3 NS3 Nrf2/DAPI ** GFPFlag NS2B3Flag 10 μm 10 μm 10 μm GFPFlag NS2B3Flag GFPFlag NS2B3Flag GFPFlag NS2B3Flag CLEC5A surface expression(gm)

9 Cheng et al. Supplemental Figure For Figure 6B NS3 For Figure 6C NS1 43 Mock 26 3 Day 5 Day 8 Day For Figure 6E 26 Mock Mock ATRA n4 n5 n4 n4 n5 n5